Dioscorealide B is an important secondary metabolite isolated from Dioscorea membranacea Pierre ex Prain & Burkill. The effect on secondary metabolite content of different concentrations of two elicitors [jasmonic acid (JA) and salicylic acid (SA)], and of medium status and JA exposure period were investigated. In the JA and SA concentration experiment, 6-week-old shoots were cultured on MS medium supplemented with 8.87 lM BA (6-benzyladenine) in combination with 100-500 lM JA or 50-200 lM SA for 3 weeks. MS medium supplemented only with 8.87 lM BA was used as a control. The highest dioscorealide B content was recorded in the 100 lM JA shoots. To determine the optimal medium status and JA exposure period, shoots were cultured on solid and in liquid MS media supplemented with 8.87 lM BA and 100 lM JA for 2, 3, 4 and 5 weeks. No interaction was found between the medium status and the elicitor exposure period in the dioscorealide B production. Shoots cultured on the solid MS medium supplemented with 100 lM JA had a higher dioscorealide B content (0.57 ± 0.35% w/w) than those cultured in liquid medium (0.36 ± 0.40% w/w) and 5-week JA exposure produced the highest dioscorealide B content of 1.05 ± 0.15% (w/w).